The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (mAbs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal antibodies, mAbs DN2 and DN6. The assay had a lower detection limit of 200pg/mL in serum and plasma samples, and showed dilution linearity and recovery ranging from 95 to 106%. The mAbs and the ELISA showed isotype specificity for PAD2. Circulating PAD2 was found in 8/28 (29%) serum samples from healthy donors. In conclusion, several of our mAbs proved useful in western blotting and immunohistochemistry, and the ELISA described here reliably measures PAD2 levels in blood. This allows investigation of PAD2 as a possible biomarker and further investigation of PAD2's involvement in various inflammatory diseases.
Journal of Immunological Methods, 2014, Vol 405, p. 15-22
Journal Article; Research Support, Non-U.S. Gov't; Animals; Antibodies, Monoclonal; Antibody Specificity; Arthritis, Rheumatoid; Biological Markers; Blotting, Western; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Humans; Hydrolases; Immunohistochemistry; Neurodegenerative Diseases; Rabbits; Reproducibility of Results; Sensitivity and Specificity; Species Specificity